The objective of this proposal is to investigate the mechanism of transcellular transport of two macromolecules, insulin and albumin, across cerebral endothelium and how infection influences such an event. During phase I, in vivo and in vitro investigation will address evidence of cerebral endothelial cell receptors for insulin and albumin. In vivo experiments will employ in situ administration of colloidal gold-ligand suspension followed by in situ brain fixation and transmission electron microscopy to assess (1) ultrastructural evidence for restricted ligand binding microdomains on the luminal membrane of cerebral microvasculature as well as evidence for ligand transcytosis; (2) physiologic evidence for saturability and competitive inhibition if specific binding sites are observed ultrastructurally. In vitro techniques will utilize isolated cerebral microvascular endothelial cells grown as a monolayer in polycarbonate filter chambers. Initial studies will address ultrastructural, immunocytochemical, and physiologic resemblance of this model to the in vivo setting. If achievable, physiologic evidence for luminal membrane ligand binding receptors will be pursued as well as vectorial transport of ligand across the monolayer. Phase II will assess the influence of infection of phase I observations by addressing (1) ultrastructural alterations in receptor density, transcytosis or development of paracellular leak of ligand during experimental meningitis (2) topographical and microvascular localization of such alterations (3) comparison of effects of live bacteria, isolated lipopolysaccharide (LPS), and interleukin-1 (IL-1) as inducers of meningitis (4) comparison of influences of LPS and IL-1 on ligand receptor binding and vectorial transport across the monolayer of cerebral endothelium.